HPV self-sampling has been widely supported by the scientific community following a strong body of literature on the subject. Self-sampling is important in cervical cancer screening as it has been shown to improve participation. It is well documented that HPV-testing has proven superior to cytology with regards to sensitivity in detection of CIN and cancer. The value of self-collected samples is reliant on the quality of the molecular testing performed, as well as the patients’ preference in sampling procedure and compliance to follow up on positive test results. Due to the incompatibility of self-samples and cytology, triage of HPV-DNA positives by testing for molecular biomarkers is highly warranted.Our objective was to compare the detection rate of genital Human Papillomavirus (HPV) infection in self- and clinician-collected samples by a 14-type HPV-DNA test and a 7-type mRNA E6/E7 test.P = 0.19). Overexpression of mRNA E6/E7 from 7 HPV types was 7.1 and 6.3%, respectively (P = 0.71). The difference between HPV-DNA and HPV-mRNA positivity rates were statistically significant in both self-collected  and clinician-collected samples . Overall agreement between the two collection methods was fair, with a concordance rate of 78.2% (390/505), k = 0.34 (95% CI: 0.25–0.44), P < 0.001, for the HPV-DNA test and 92.5% (467/505), k = 0.40 , P < 0.001, for the mRNA test, respectively. 96.8% of the participants reported they felt confident carrying out the self-collection themselves, and 88.8% reported no discomfort at all performing the procedure.Five hundred five women were recruited. Each study participant had two sample collection procedures performed upon the same visit, alternating order in execution of the self-collection or the clinician-taken procedure first or second, 1010 samples in total. HPV-DNA prevalence was 22.8% in self-collected versus 19.2% in clinician-collected samples (This comparative study of two sampling methods reports fair agreement of HPV positivity rates between the self-collected and clinician-collected specimens using Abbott hrHPV and PreTect HPV-Proofer’7 tests. Only one third of HPV-DNA positive women had overexpression of mRNA E6/E7.ISRCTN77337300.The online version contains supplementary material available at 10.1186/s12879-021-06189-2. Cervical cancer is a major public health problem, and the second most common cancer in women living in less developed regions with an estimated 570,000 new cases in 2018. According to the World Health Organization, approximately 311,000 women died from cervical cancer in 2018: more than 85% of these deaths occurring in low- and middle-income countries , 5 and wCurrently, most guidelines recommend cytology in triage of HPV-DNA positives. As PAP reflex testing is not applicable on self-collected material, a new clinician-collected sample is required for the purpose of triage, hereby suffering increased risk of loss to follow up; based on the nature of non-attenders. An alternative to cytology in triage could be detection of molecular biomarkers; being compatible on self-sampled material .Literature describes effects caused by device and specimen processing in terms of various concentration and quality of cellular material that possibly impair test performance , 13 whilThis study was conducted to compare the performance of self-collected samples by using a self-sampling device  versus clinician-collected samples by using the professional-use device  in combination with a 14-type HPV-DNA and 7-type mRNA E6/E7 test as the primary outcome.Patient-reported acceptability of the self-collected samples method was the only secondary outcome.The study was approved by the institutional ethics review board (CI/243/18) at Eduardo Liceaga, Mexico General Hospital, Mexico City, before commencing.http://www.isrctn.com/ISRCTN77337300Trial registration: ISRCTN, ISRCTN77337300. Registered 15 December 2020 - Retrospectively registered, During the period August 2018 to April 2019, study participants were recruited among women attending cervical cancer screening and among health professionals working at the Oncology clinic of Eduardo Liceaga, Mexico General Hospital, Mexico City. Eligible were sexually active women aged 30–65 that responded positively to the invitation and had no history of medical or surgical treatment  for cervical cancer. Excluded were pregnant and breastfeeding women, those who had had sexual activity within 24 h before to the collecting samples procedure, and those who chose not to sign the informed consent. Prior to enrollment, the purpose and nature of the study were explained to each participant to obtain their written consent. The two sampling procedures were performed upon the same day, in alternating order for each participant upon arrival. Since the study was conducted at the clinic for cervical screening, clinicians’ additional training was unnecessary to collect the reference sample. For the self-sampling device, the manufacturer’s written and illustrated instructions for use in Spanish were provided to all participants. The procedure was performed in a separate room for the women’s discretion without assistance by health staff. Finally, after the samples were collected, the participant completed a questionnaire about her experience during the self-collection procedure. Respondents scored items on an 8-point Likert scale, ranging from 1 (“no discomfort”) to 8 (unbearable discomfort”). All women were informed to discuss their test results upon completion with a healthcare professional to decide on further follow-up. Patient sensitive information was anonymized by a 6-digit code and registered using Microsoft Excel 16.16.23 (200615–2016). Only study responsible staff at the hospital had access to the registered information.The participants of this study performed self-collection of cervicovaginal material using the XytoTest medical device , an ergonomically designed device to be inserted into the vagina, with a diameter less than 8 mm and a length of 14 cm [A clinician collected the sample by using the Cervex-Brush® , which is among the most widely used method for cervical cancer screening, commonly used as a reference method for validation of new devices . After sme HR HPV test  is an automated, qualitative multiplex assay based on real-time polymerase chain reaction (PCR) intended to detect 14 high-risk HPV genotypes  and to partially genotype 16, 18 from the other 12 high risk genotypes. Human Beta-globin is detected as an internal control. The test has been clinically validated according to international consensus guidelines and proved to also be accurate for self-collected samples [The Abbott RealTi samples , 18. AllPreTect HPV-Proofer’7  is a diagnostic kit for the qualitative detection and direct typing of E6/E7 mRNA from HPV 16, 18, 31, 33, 45, 52 and, 58. The kit contains an intrinsic sample control (ISC) targeting a human housekeeping gene to assess specimen quality and reveal possible factors that may inhibit amplification. The kit utilizes real-time NASBA technology, an enzymatic one-step amplification process able to amplify RNA under isothermal conditions at 41 °C . SeveralThe sample size was estimated considering a two-tailed hypothesis, with a 95% confidence interval, a statistical power of 80%, and an effect size (Cohen’s d) of 0.12555. With these characteristics, the estimated sample size required was 500 study participants, calculated using the software GPower 3.1.9.2. Microsoft Excel 2016  and IBM SPSS Statistics software package version 21  were used for data collection and evaluation. Cohen’s Kappa coefficients were calculated to evaluate the agreement between the two sampling methods regarding hr-HPV results, applying the most commonly used scale to express the strength of the agreement as follows: 0.00–0.20, 0.21–0.40, 0.41–0.60, 0.61–0.80 and ≥ 0.81 indicated slight, fair, moderate, substantial, and almost perfect, respectively. The Wilcoxon signed -rank test was applied to evaluate the differences between the two sampling methods. A statistically significant difference was defined as a 5% chance of a type I error (α ≤0.05). For comparison of the presence of HPV-16, HPV-18, and 12 other hr-HPV genotypes between the two samples, the following terminology was used: concordant or discordant. A concordant result was determined if results from all three channels  showed at least one identical genotype in both samples, where discordance represented no similarities in genotypes at all.Overall, a total of 505 women participated in the study, everyone within the inclusion criteria. 65.5% of the participants were recruited among women attending the general screening program at the clinic, and 33.5% were recruited among the health professionals working at the Mexico General Hospital. The inclusion of health professionals was partially to learn whether the level of education might affect the quality of the sample evaluated by cellularity and sample integrity if the instructions for self-collection was not sufficiently clearly described as health professionals presumably would have a better understanding of sample taking in general. In total 1010 samples were collected equally distributed between self-collected samples, and clinician collected specimens. All the collected samples by the two procedures were valid for processing. The mean age of the participants was 43.8 ± 8.1 years . Just over two-thirds of the women (69.7%) reported they were older than 18 years when they first had sexual intercourse, and less than a third (30.3%) were under the age of 18 , whilst the positivity rate for HPV mRNA E6/E7 was about one third; 7.1%  versus 6.3%,  respectively (P = 0.71).The prevalence of hr-HPV-DNA varied from 22.8%  to 19.2%  among the self-collected and clinician-collected samples (P > 0.05). Mostly non-HPV16/18 genotypes were detected. The vast majority of the infections identified in self-collected samples were single (85.2%) and multiple infections were found in 17 specimens (14.8%) in line with 84.5 and 15.5% among clinician-collected samples , k = 0.40 (95% CI: 0.25–0.56), P < 0.001, for the HPV mRNA test, respectively. In women with at least one positive HPV-test, the agreement was 31.7% (51/161) for the HPV-DNA test and 28.3% (15/53) for the HPV mRNA test. Kappa for the mRNA test in the 161 women with at least one positive HPV DNA test was 0.30 (95% CI: 0.12–0.48), P < 0.001  had discordant HPV-DNA results. In 46 cases, the women were clinician-collected sample HPV-positive/self-collected sample HPV-negative. Among these, 24 had the clinician-collected sample taken first and 22 women the self-sampling first in order. Sixty-four women were clinician-collected sample HPV-negative/self-collected sample HPV-positive; among those of whom 27 had their first sample by their clinician versus 37 women self-sampling first. Five women were detected as hr-HPV positive in both samples collected, with no similarities in genotypes. Regardless of order of the method used, no significant differences in the agreement rates for self-collected, and clinician-collected sampling techniques were observed, P < 0.001), tested using Wilcoxon signed rank test.All 505 paired samples obtained valid results for the two subsequent HPV amplification tests done, hence no exclusions were made due to low cellularity. The manual cell count using Bürker chamber was done for a random subset of paired samples to evaluate the number of cells collected by each sampling method, 97 pairs in total. Forty-seven of the self-collected samples were done by health professionals, 50 self-collected by women attending screening. The average number of cells per milliliter was calculated following standard protocol and descriptive statistics are presented in Table . The selP = 0.19).Evaluating the cellularity obtained by self-collection performed by the two subgroups of study participants , no difference was observed  is designed to optimize clinical performance, and since no relevant clinical outcome by the means of cytology or histology is available in this study, this will not be possible to discuss. It is well known that PAP test sensitivity  varies dWithin this study population, the hr-HPV detection rate by PCR assay was about 20%, which is comparable to the prevalence (24.7%) found in another study conducted in the Mexican population . ClinicaThe obtained significant higher cellularity per milliliter for the self-collected versus the clinician-collected specimens in this study might be explained by the fact that XytoTest has an area coated with a highly adhesive hypoallergenic elastomer and therefore cells are more readily collected as soon as the device is inserted into the vagina.It is known that molecular biomarkers are of vital importance in relation to the triage of patients in cervical screening programmes . All curThe perspective of implementing HPV mRNA in triage of test positives are among one of the options discussed to represent an improved, highly specific risk-based approach for maximizing screening benefits and minimizing harms –21, 30.Our data confirm 7-type mRNA testing to be applicable for reflex testing on self-collected specimens. Among the study participants, only one third of the HPV infected women had a positive mRNA test, an appealing situation for effective triage reducing the number of colposcopies and biopsies.Recent research has proven the 7 genotypes included in the mRNA test to be the most important to screen for, being responsible for 90% of all cervical cancers , 31, 32.Reliable self-sampling methods and molecular diagnostics may significantly aid prevention of cervical cancer, by simplicity, increased accessibility to screening and accurate diagnostics.By combining HPV-DNA testing and identification of mRNA E6/E7 biomarkers, both high sensitivity and specificity might be maintained for improved patient management , 34.A clear limitation of this study is the lack of clinical data; cytology and a histology examination of samples that were found to be hr-HPV positive or mRNA E6/E7 positive, which would have enabled associations to be drawn between molecular assays with possible morphological changes, and presumably identify progression of cervical lesions. Unfortunately, these data were not available for our review, nor contemplated in the inclusion criteria of this study.Although the acceptability of self-collected samples was found to be high among the women participating in this study, the questionnaires are of limited reliability because the study administered no parallel healthcare provider-reported questionnaire.This comparative study of two sampling methods reports fair agreement of hr-HPV positivity rates between the self-collected and clinician-collected specimens using Abbott hr-HPV and PreTect HPV-Proofer’7 tests. Only one third of HPV-DNA positive women had overexpression of mRNA E6/E7, effectively discriminating women warranted for immediate colposcopy/biopsy from return to follow-up and suggests longer follow-up interval for single HPV-DNA positive women. Such a strategy will inevitably reduce over-referral for colposcopy but needs clinical and cost-benefit assessment in prospective studies.Additional file 1.


Coronavirus Disease 2019 is a primarily respiratory illness that can cause thrombotic disorders. Elevation of D-dimer is a potential biomarker for poor prognosis in COVID-19, though optimal cutoff value for D-dimer to predict mortality has not yet been established. This study aims to assess the accuracy of admission D-dimer in the prognosis of COVID-19 and to establish the optimal cutoff D-dimer value to predict hospital mortality.Clinical and laboratory parameters and outcomes of confirmed COVID-19 cases admitted to four hospitals in Kathmandu were retrospectively analyzed. Admitted COVID-19 cases with recorded D-dimer and definitive outcomes were included consecutively. D-dimer was measured using immunofluorescence assay and reported in Fibrinogen Equivalent Unit (μg/ml). The receiver operating characteristic curve was used to determine the accuracy of D-dimer in predicting mortality, and to calculate the optimal cutoff value, based on which patients were divided into two groups and predictive value of D-dimer for mortality was measured.182 patients were included in the study out of which 34(18.7%) died during the hospital stay. The mean admission D-dimer among surviving patients was 1.067 μg/ml (±1.705 μg/ml), whereas that among patients who died was 3.208 μg/ml (±2.613 μg/ml). ROC curve for D-dimer and mortality gave an area under the curve of 0.807 . Optimal cutoff value for D-dimer was 1.5 μg/ml . On Cox proportional hazards regression analysis, the unadjusted hazard ratio for high D-dimer was 6.809 , and 5.862  when adjusted for age.D-dimer value on admission is an accurate biomarker for predicting mortality in patients with COVID-19. 1.5 μg/ml is the optimal cutoff value of admission D-dimer for predicting mortality in COVID-19 patients. Coronavirus disease 2019 (COVID-19), caused by the Severe acute respiratory syndrome coronavirus 2, was first recorded in Wuhan, the capital of Hubei province of China in December 2019 . ThromboD-dimer is a fibrin degradation product, widely used as a biomarker for thrombotic disorders. A D-dimer value less than 0.5 μg/mL is usually considered normal, and values increase with increasing age and in pregnancy. The level of D-dimer rises with increased severity of community-acquired pneumonia –9.Accurate and widely available prognostic biomarkers can be very useful in the management of COVID-19. This multi-center study aims to assess elevation in D-dimer at the time of admission as a possible prognostic indicator of mortality in COVID-19 patients. The cutoff value used for D-dimer shows significant variation between the published studies, and there seems to be no consensus yet on what the best cutoff value is to predict severity or mortality. We thus aim to establish the optimal cutoff value for D-dimer that can be used clinically for predicting mortality in COVID-19 patients.This retrospective study was conducted in four different tertiary care centers in the capital of Nepal, each of which were designated hospitals for COVID-19 patients. Ethical approval was granted by the Nepal Health Research Council Ethical Review Board. Permission and approval were taken from each hospital prior to the initiation of the study. Standards for Reporting Diagnostic accuracy studies (STARD) 2015 reporting guidelines were followed.The sample size was calculated using the methodology described by Buderer, with an expected sensitivity of 92.3% and an expected specificity of 83.3% taken from a reference study . The mor2) less than 94% and symptomatic cases were consecutively enrolled in the study. Exclusion criteria included cases without recorded D-dimer values at admission, presence of other infections, prior anticoagulant use, and deep vein thrombosis/pulmonary embolism (DVT/PE), and cases without recorded definitive outcomes .Adults (aged 18 years or older) diagnosed with COVID-19 by Reverse transcription polymerase chain reaction (RT-PCR) and admitted to one of the four study centers between March 1, 2020, and December 31, 2020 were screened for inclusion in the study. Asymptomatic cases with Peripheral oxygen saturation  or non-survival . The secondary outcome variable was the total duration of hospital stay.All demographic, clinical, and outcome data were extracted from the patients’ hospital record files. Demographic characteristics of patients , D-dimer on admission, SpOBlood samples for D-dimer assessment were collected within 24 hours of admission and sent to the respective hospital laboratories. All measurements were done in the laboratories within 2 hours of sample collection.D-dimer was measured by immunofluorescence using kits from different manufacturers in different centers. The different analyzers used were STANDARD F200 Analyzer , POCT Axceed P200 (Bioscience (Tianjin) Diagnostic Technology Co., Ltd), Getein 1100 Immunofluorescence Quantitative Analyzer , and mispa-i2 . All the kits used had a biological reference range of <0.5 μg/ml, and all results were reported in Fibrinogen Equivalent Units .All cases received low molecular weight heparin (LMWH), steroids, and remdesivir unless contraindicated.Continuous variables were expressed as mean ± standard deviation. Categorical variables were presented as n (%). The difference between the two cohorts was determined using the independent two-sample T-test for continuous variables. For categorical variables, Fisher’s exact test was used when one or more cells in the contingency table had counts of less than 5. A chi-squared test was used for other categorical variables. Duration of hospital stay between groups was compared using the Mann-Whitney U test. The accuracy of D-dimer as a predictor of mortality was calculated using the receiver operator characteristics (ROC) curve. The area under the curve, sensitivity, and specificity was calculated from the ROC curve. The optimal cutoff value for D-dimer was determined by the value corresponding to the point in the curve closest to the top-left of the ROC graph. Based on this cut-off value, patients were categorized into two groups. The 30-day outcomes of these two groups were compared using Kaplan-Meier survival analysis. Cox proportional hazards regression analysis was performed with a 95% confidence interval. Univariate analysis was performed first and only variables showing significant results in univariate analysis were included in calculating the adjusted hazard ratio. The software used for statistical analysis was Statistical Package for Social Sciences (SPSS) version 26. A p-value less than 0.05 was considered statistically significant.A total of 182 eligible candidates were enrolled in the study, among which 113 (62.1%) were male and 69 (37.9%) were female. The mean age of enrolled participants was 58.16 years (±15.65 years).34 (18.7%) patients died during hospital stay. One patient was discharged on the patient’s request and one patient left against medical advice. The area under the curve for receiver operating characteristic (ROC) curve for D-dimer values on admission against patient outcome  was 0.80Based on the cutoff value of 1.5 μg/ml, all patients were categorized into two groups for comparison, summarized in The mean D-dimer values on admission by age and sex are summarized in 126 out of 182 patients had D-dimer on admission less than 1.5 μg/ml, out of which 10 patients (7.9%) died during hospital stay and 116 were discharged (including one discharge on patient request). Of the 56 patients who, on admission, had D-dimer of 1.5 μg/ml and above, 24 (42.9%) died and 32 were discharged .The mean admission D-dimer among patients who survived was 1.067 μg/ml (±1.705 μg/ml), whereas that among patients who died was 3.208 μg/ml (±2.613 μg/ml). This is a highly statistically significant difference .Subgroup analysis of male and female patients showed different optimal cutoff values; 1.90 (0.759–0.94) with sensitivity of 71.4% and specificity of 90.2% for males and 1.46 0.583–0.880) with sensitivity of 69.2% and specificity of 66.1% for females . Among the 146 patients who recovered and were discharged routinely , 113 had D-dimer at the time of admission of less than 1.5 μg/ml, while 31 had D-dimer on admission higher than 1.5 μg/ml. A separate analysis of these patients, who were discharged routinely, included in 30-day survival of patients was analyzed using the Kaplan-Meier procedure, which showed a significant difference (Log-rank (Mantel-Cox) of 0.00) in survival between the low D-dimer and high D-dimer groups at 30 days .The results of univariate Cox regression, as shown in Further analysis among the deceased cases was done taking into consideration the laboratory parameters including hemoglobin, WBC with lymphocyte, platelet count, C- reactive protein, prothrombin time (PT) and activated partial thromboplastin time (aPTT) as shown in Patients received plasma therapy, non-invasive and invasive ventilation as per requirement.Similarly, the immediate cause of death was analyzed among the both high and low D- dimer groups, as shown in The current study included 182 cases admitted to the hospital with a diagnosis of COVID-19 from March to December 2020, which was in the relatively early phase of the pandemic in Nepal. Cases were managed in the hospital-based on available resources and guidelines, the latter of which were not established early on and evolved with time. D-dimer was usually measured on admission, and serial D-dimer measurement was not part of routine management. Treatment during the study period was largely symptomatic, consisting of antipyretics, analgesics, and supplemental oxygen when required. LMWH was given prophylactically to all patients without contraindications.This study found that a higher D-dimer value on hospital admission was significantly associated with in-hospital mortality in patients of COVID-19. D-dimer is a fibrin degradation product and its main utility is in the diagnosis and management of thrombotic disorders. Before the 2019 COVID-19 pandemic, D-dimer was not considered a useful biomarker for bacterial or viral pneumonia despite some evidence to the contrary . Since tA systematic review by Rostami et al. reported the mean D-dimer level to be 0.58 μg/ml in 1551 patients with mild disease and 3.55 μg/ml in 708 patients with severe disease . A meta-The area under the curve (AUC) of the ROC curve for D-dimer on admission in our study was 0.807. An area under the curve of more than 0.8 is generally considered to indicate ‘good accuracy’ of the test, whereas values above 0.7 are considered to indicate ‘fair accuracy’. This finding is in accordance with most of the published research on the topic. Zhang et al found an AUC of 0.89 in their study, whereas Oualim et al reported an AUC of 0.775 and Peiro et al. reported an AUC of 0.756 , 18. OurThe optimal cutoff value for admission D-dimer for predicting mortality has not been agreed upon in the current literature. Some studies have identified optimal cutoffs based on ROC curves, but the values range anywhere from 0.67 to 2.025 μg/ml, with large variation in sensitivity and specificity as well , 17–19. A recent study found that the trajectory of lab values including D-dimer in hospitalized patients had good accuracy in predicting mortality and severity of COVID-19 . ConsideThere is significant heterogeneity among studies on D-dimer and COVID-19. Different laboratories use different kits for measurement and the accuracy and reliability of measurement can vary according to the kit manufacturer. Furthermore, there is variation in reporting units. Favaloro and Thachil analyzed 20 papers on COVID-19 and D-dimer and found that most papers did not report which manufacturer and reagent kit was used and whether D-dimer values were reported in D-dimer units (DDU) or Fibrinogen equivalent units (FEU). They also found that nearly half the studies did not report normal cutoff values . This laThe most common reason cited in the literature for the elevation of D-dimer includes viremia and the cytokine storm syndrome, in which the rise in pro-inflammatory cytokines  are inadequately controlled by the anti-inflammatory factors which overwhelm the coagulation cascade . HypoxiaA major limitation of our study is selection bias because of its retrospective nature. Only patients admitted to the hospital were included, which meant that asymptomatic patients with high oxygen saturation, who were not admitted according to hospital guidelines, were not included in the study. Some otherwise eligible cases also had to be excluded due to incomplete laboratory tests and medical records, specifically D-dimer on admission. Time from illness onset to hospital presentation may affect the D-dimer values. Since the study was conducted in four centers with their own laboratories, different kits were used for the measurement of D-dimer in different centers, which causes the potential for measurement bias due to the use of different equipment. The reference ranges of D-dimer and the units of reporting are the same for all four centers, which should mitigate this to some extent.D-dimer is a widely available, relatively inexpensive, and easy to perform laboratory test, and our study found that it has good accuracy in predicting in-hospital mortality in COVID-19 patients. It can be used as a metric for identifying high-risk cases and can assist in choosing appropriate management. Incorporation of D-dimer into routine investigation and risk assessment of COVID-19 patients can prove useful in tackling this global health challenge.D-dimer value on admission is an accurate biomarker for predicting mortality in patients with COVID-19 and 1.5 μg/ml is the optimal cutoff value of admission D-dimer for predicting mortality in COVID-19 patients, with good sensitivity and specificity. D-dimer can thus be an easy to perform and inexpensive laboratory indicator for COVID-19 prognosis.S1 File(SAV)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.


Dear Editor,1, are the two most fundamental nutrients for all living organisms. As one of the most ancient autotrophic bacteria, cyanobacteria utilize photosynthesis to convert the inorganic carbon (Ci) into carbohydrates. Carbon fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which is a naturally inefficient enzyme2. In response to gradually decreased CO2 and elevated O2 levels in the atmosphere, cyanobacteria have evolved a unique CO2-concentrating mechanism (CCM), which can substantially accumulate CO2 in the vicinity of RuBisCO for improved carboxylation efficiency3. The cyanobacterial CCM consists of a subcellular self-assembled icosahedral microcompartment, termed the carboxysome, and several Ci uptake systems3. To date, five Ci-uptake systems have been identified in cyanobacteria, including three bicarbonate transporters BicA, SbtA, and BCT1, in addition to two CO2-uptake complexes: NDH-I3 and NDH-I4.Carbon and nitrogen, the uptake and intracellular metabolisms of which are tightly coupled3– symporter within the TC.2.A.83 family of Na+/solute symporters5. At a low level of intracellular Ci, SbtA of an up-regulated level can recruit its partner protein SbtB to the membrane6. The sbtB gene from the sbtA–sbtB operon encodes a PII-like signaling protein that senses the intracellular level of the secondary messenger cAMP that correlates with high Ci conditions, versus AMP at low Ci conditions, thus functioning as a Ci sensor in cyanobacteria6.SbtA, which is ubiquitous in cyanobacteria, is an inducible high-affinity sodium-dependent HCOSynechocystis sp. PCC 6803 SbtA and solved its cryo-EM structure at 3.50 Å resolution , they form a complex9. To elucidate the fine interaction mode between SbtA and SbtB, we purified the SbtA–SbtB complex in the presence of AMP and solved its 3.15 Å cryo-EM structure (Supplementary Fig. 10, SbtB also adopts a trimeric structure, each subunit of which shows a canonical PII ferredoxin-like fold with a well-structured T-loop (Fig. It has been reported that when SbtB and SbtA are co-expressed in Escherichia coli (oop Fig. . The C-toop Fig. . In addioop Fig. .2. The inter-domain cleft of SbtA is formed by the intracellular parts of TM2 and TM7 of the gate domain, in addition to TM9 and TM10 of the core domain. The extended SbtB T-loop runs along the hydrophobic inter-domain cleft of SbtA, forming extensive hydrophobic interactions (Fig. –GlnK, in which the T-loop of PII protein GlnK inserts deeply into the cytoplasmic pore exit of the ammonia channel AmtB11, the SbtA–SbtB structure showed that SbtB T-loop partially blocks the substrate tunnel exit of SbtA, indicating that SbtB might attenuate the transport activity of SbtA under certain physiological conditions. Consistently, the previous results showed that SbtB inhibited the SbtA-mediated HCO3– uptake in E. coli9. However, a recent report demonstrated that the light-regulated SbtA function in vivo is independent of SbtB in Synechococcus PCC 794212. Thus the fine in vivo regulatory mechanism of SbtA–SbtB remains unsolved, but might be involved in the coordinated regulation of light–dark transitions, the intracellular homeostasis of adenyl-nucleotides and redox status.In the complex, the core structure of SbtB does not directly interact with the intracellular surface of SbtA. Instead, the structured T-loop of SbtB, which extends ~20 Å from the core structure of SbtB, is inserted into the inter-domain cleft of the corresponding SbtA subunit Fig. , yieldinons Fig.  via relaons Fig. . Similar–SbtB complex, there is a clear cryo-EM density most likely corresponding to a sodium ion (Fig. +-dependent transporter. In each subunit, the Na+ forms five coordinate bonds with the main-chain oxygens of residues Phe110, Gly111, and Ala112 from TM4a and Ala320 and Ser322 from TM9a (Fig. +-dependent transporters13, Na+ and its co-transported substrate are located nearby the crossover of TMs. We thus docked HCO3– in the vicinity of the crossover of the complexed SbtA via HADDOCK14. In this docking model, the HCO3–-binding site is located at the interface of the core and gate domains. It binds to a cavity formed by the other side of the crossover and forms four hydrogen bonds with Ser114, Tyr140, Ser324, and Ser327 (Fig. 3–-binding residues to Ala led to a dramatic decrease of HCO3– transport activity of SbtA in E. coli membrane vesicles to about 10%–40% of the wild-type level (Fig. At the peripheral side of TM crossover within the center of SbtA core domain in the SbtAion Fig. , given SM9a Fig. . Notably327 Fig. . Sequenc327 Fig. . Furthervel Fig. . The res3– transport (Fig. 3– and Na+. Substrate binding most likely promotes SbtA to adopt an occluded conformation, as seen in our free SbtA structure with a Na+ bound in the pocket. Afterwards, the core domains of SbtA slide toward the cytosolic side against the immobile gate domains, resulting in an inward-open conformation of SbtA. Accordingly, the substrate tunnel is open toward the intracellular space (Supplementary Fig. 15.Structural analysis revealed that the substrate-binding sites of free SbtA are inaccessible to the solvent from either intracellular or extracellular side. Thus, the structure of free SbtA represents an occluded conformation. Respectively superimposing the two individual domains of each SbtA subunit against those in the SbtB-bound form showed little structural variations (Supplementary Fig. ort Fig. . Initial3– transporter SbtA, which exhibits a new architecture of Na+/solute symporters and its fine interaction pattern with a PII-like protein SbtB. Structural analysis combined with site-directed mutagenesis enabled us to identify the substrate-binding pocket and propose an elevator transport mechanism of SbtA.In summary, this study provides structural insights into the cyanobacterial high-affinity HCOSupplementary Information


Trypanosoma cruzi infection and remains a relevant cause of chronic heart failure in Latin America. The pharmacological arsenal for Chagas disease is limited, and the available anti-T. cruzi drugs are not effective when administered during the chronic phase. Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) have the potential to accelerate the process of drug discovery for Chagas disease, through predictive preclinical assays in target human cells. Here, we aimed to establish a novel high-content screening- (HCS-) based method using hiPSC-CMs to simultaneously evaluate anti-T. cruzi activity and cardiotoxicity of chemical compounds. To provide proof-of-concept data, the reference drug benznidazole and three compounds with known anti-T. cruzi activity (a betulinic acid derivative named BA5 and two thiazolidinone compounds named GT5A and GT5B) were evaluated in the assay. hiPSC-CMs were infected with T. cruzi and incubated for 48 h with serial dilutions of the compounds for determination of EC50 and CC50 values. Automated multiparametric analyses were performed using an automated high-content imaging system. Sublethal toxicity measurements were evaluated through morphological measurements related to the integrity of the cytoskeleton by phalloidin staining, nuclear score by Hoechst 33342 staining, mitochondria score following MitoTracker staining, and quantification of NT-pro-BNP, a peptide released upon mechanical myocardial stress. The compounds showed EC50 values for anti-T. cruzi activity similar to those previously described for other cell types, and GT5B showed a pronounced trypanocidal activity in hiPSC-CMs. Sublethal changes in cytoskeletal and nucleus scores correlated with NT-pro-BNP levels in the culture supernatant. Mitochondrial score changes were associated with increased cytotoxicity. The assay was feasible and allowed rapid assessment of anti-T. cruzi action of the compounds, in addition to cardiotoxicity parameters. The utilization of hiPSC-CMs in the drug development workflow for Chagas disease may help in the identification of novel compounds.Chagas disease is caused by  Trypanosoma cruzi, affects approximately 6 to 7 million people worldwide [Chagas disease, caused by the hemoflagellate protozoan orldwide , 2. Duriorldwide , 4.in vitro screening process of anti-T. cruzi may contribute to accelerate the drug discovery process.Currently, benznidazole and nifurtimox are the only medications available to treat Chagas disease, both with proven efficacy when administered during the acute phase of the disease, which is often underdiagnosed . In addiIn the past years, human-induced pluripotent stem cells (hiPSCs) have contributed to drug discovery and toxicological studies applied to cardiovascular diseases , 8. hiPSin vitro drug discovery method using T. cruzi-infected hiPSC-CMs and multiparameter analyses using a high-content screening (HCS) platform.Unpredicted cardiotoxicity is one of the main causes of drug withdrawal from the market and is the result of the low predictive value of currently available methods for preclinical cardiac toxicity testing . This caCell reprogramming and experiments with hiPSCs received approval from the Ethics and Research Committee (IRB) at São Rafael Hospital (CAAE 20032313.6.0000.0048).We used two hiPSC lines obtained from two donors, previously obtained by integration-free reprogramming of erythroblasts with episomal vectors . The celμM Y27632  was added to the culture medium. On Days -2 and -1, the hiPSC medium was exchanged, and on Day 0, mTeSR1 was replaced by Cardiomyocyte Differentiation Medium A, followed by Cardiomyocyte Differentiation Medium B on Day 2 and Cardiomyocyte Maintenance Medium on Day 4. The medium was then renewed every two days with Cardiomyocyte Maintenance Medium. Spontaneous beating clusters began to be observed on protocol Day 8.hiPSCs were differentiated into cardiomyocytes using the PSC Cardiomyocyte Differentiation Kit . Briefly, the hiPSCs were dissociated with ReleSR  into a single cell suspension and replated at a 1 : 8 ratio in 12-well Matrigel-coated wells, being cultured with mTeSR1 . During the first 24 h after plating (Day -3), 10 For immunofluorescence, the cells were fixed with 4% paraformaldehyde (PFA), washed with PBS, and permeabilized with 0.05% Triton solution. The following primary antibodies and dilutions were used, with overnight incubation at 4°C: sarcomeric alpha-actinin , MF20 , anti-cTNT , and GATA-4 . The cells were incubated with the secondary antibodies, for 1 h at room temperature with anti-mouse IgG Alexa Fluor 568-conjugated or anti-rabbit IgG Alexa Fluor 488-conjugated, both diluted at 1 : 500 (Thermo Fisher Scientific). Images were captured using a confocal microscope .μg of RNA, according to the manufacturer's recommendations. Synthesis of cDNA and RNA expression analysis was performed by Real-Time PCR using TaqMan Gene Expression Assay for GATA4 (Hs 01034628_m1), NKX2.5 (Hs 00231763_m1), and TNNT2 (Hs 00943911_m1). All reactions were run in duplicate on an ABI 7500 Real-Time PCR System (Applied Biosystems) under standard thermal cycling conditions. A nontemplate control (NTC) and nonreverse transcription controls (No-RT) were also included. The samples were normalized with GAPDH (endogenous control). The threshold cycle (2 − ΔΔCt) method of comparative PCR was used to analyze the results [RNA was extracted from the cells with TRIzol reagent  and the concentration determined by photometric measurement. A High-Capacity cDNA Reverse Transcription Kit  was used to synthesize cDNA from 1  results . Data waThe cells were dissociated into single-cell suspensions by incubation with trypsin-EDTA solution , at 37°C for 5 min, followed by centrifugation at 350 g for 5 min at room temperature. The cells were counted and stained with an APC-conjugated anti-TRA1-60 antibody  for pluripotency evaluation. The efficiency of cardiac differentiation was evaluated by staining with anticardiac troponin T , after permeabilization with 0.3% Triton X-100 solution . The secondary antibody anti-mouse IgG conjugated with Alexa Fluor 647 was then used . Data acquisition was performed with the LSR Fortessa flow cytometer  using the FACSDiva v.6.3 acquisition and analysis software.T. cruzi. Trypomastigotes were obtained from T. cruzi-infected BALB/c mice at 7 d.p.i., as previously described [in vitro by infecting LLC-MK2 cells (ATCC CCL-7). The Animal Ethics Committee at Gonçalo Moniz Institute, Fiocruz, approved this protocol under the number 17/2017. The cells were cultured in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin   and incubated at 37°C and 5% CO2 for 7 days before harvesting of free trypomastigote forms in the supernatant.Infection of cardiomyocytes was performed using the Y strain escribed  and mainT. cruzi infection experiments were performed with hiPSC-CMs obtained from three donors, two generated in our laboratory and the third one commercially obtained . The characterization of commercially obtained hiPSC-CMs was previously published [4 cells/well) were plated in 96-well plates coated with Matrigel  and kept at 37°C and 5% CO2 for 24 h. Then, the cells were infected with 1 × 105 or 2 × 105 trypomastigotes/well (multiplicity of infection (MOI) 5 or 10, respectively). The assay was adapted to 384-well plates, using 7 × 103 cells/well and infection with 3.5 × 104 trypomastigotes/well (MOI 5) for 24 h. On the following day, the wells were washed, and fresh media was added with the selected compounds.ublished . hiPSC-CT. cruzi compound, was used for comparison. Other molecules—doxorubicin and endothelin-1 —were used in the nonlethal toxicity standardization assay.Three compounds endowed with anti-T. cruzi activity previously determined in conventional assays were tested: a betulinic acid derivative named BA5 and two thiazolidinone compounds named GT5A and GT5B –22. The 4 cells/well and 7 × 103 cells/well, respectively, and infected for 24 h, as described in the previous section. After incubation with the compounds at different concentrations for 48 h, the cells were fixed with 4% PFA and labeled with Hoechst 33342  or DRAQ5 . The images were acquired with the Operetta High Content System . Nuclei were delimited by the Hoechst 33342 or DRAQ5 (segmentation) channel using the Harmony software. The cytoplasm was segmented through the Alexa 594 channel, corresponding to the troponin T labeling. Intracellular amastigotes were detected as spots stained with DRAQ5 in the cytoplasm. Mock-infected control cells were used to exclude other nonspecific cytoplasmic spots from the analysis, by using morphological and fluorescence intensity parameters for selection. The experiments were carried out in triplicate for each test condition. Following cell segmentation and parameter selection, the total number of cells, the number of infected cells, the total number of amastigotes, the number of amastigotes per cell, and the infection rate were calculated. The cytotoxic concentration for 50% of the cardiomyocyte population (CC50), the inhibitory concentration for 50% of the amastigote population (EC50), and the selectivity index were calculated for test compounds and standard drug (benznidazole). The calculation of nonlinear regression to obtain the EC50 value was evaluated using Prism 7.04 .hiPSC-CMs were plated in 96- or 384-well plates at densities of 2 × 10The cytotoxicity of the compounds was evaluated by counting the number of cells in each well  and by evaluating other parameters of nonlethal toxicity: cell and nuclear morphology, damage to the cytoskeleton using Phalloidin-488 staining, and mitochondria biomass using MitoTracker Red staining . The parameters were plotted in a principal component panel, where the drugs are clustered, and it was observed what groups were closer to control groups, after 48 hours of treatment. The parameters used in the evaluation of the cytoskeleton score were area, roundness, fluorescent intensity, and coefficient of variation (CV) of intensity to assess homogeneity of markers in the cell. To evaluate mitochondria biomass, we used fluorescent intensity, CV of intensity, the texture index SER “Hole” and texture index SER “Saddle,” and the texture-based analysis, to access the pixel intensity, showing the effects of the drugs on mitochondrial morphology Figure .To evaluate cardiomyocyte mechanical stress and hypertrophy processes, the concentrations of the N-terminal prohormone of brain natriuretic peptide (NT-pro-BNP) in the cardiomyocyte culture supernatants were evaluated. Culture supernatants were collected 48 hours after incubation with the compounds, pooled, and frozen at -80°C until analysis with the commercially available kit Vidas® NT-pro-BNP , following the manufacturer's recommendations.t-test. Nonparametric data were assessed using the Mann–Whitney test. For comparison between three or more groups, the ANOVA test with Tukey's posttest for parametric data and the Kruskal-Wallis with Dunn's posttest for nonparametric data were used. Values of p < 0.05 were considered statistically significant. The EC50 values were obtained through nonlinear regression analyses and the selectivity index by dividing the average of the CC50 values over EC50 (IS = CC50 ÷ EC50). Correlations between continuous variables were evaluated by the Pearson or Spearman coefficients.Parametric data were evaluated using Student's T. cruzi infection and to define the optimal MOI to be used in the assay, a preliminary test was performed using MOIs 5 and 10. hiPSC-CMs were highly permissive to T. cruzi infection, leading to similar infection rates . The average number of amastigotes per cell was significantly higher for MOI 10 compared to 5   . A nonstctively) . MOI 5 wInfected hiPSC-CMs were treated with the standard drug, benznidazole, in different concentrations. Benznidazole was effective in reducing the percentage of infection in a concentration-dependent manner . None ofT. cruzi activity of two synthetic and one semisynthetic compounds , which were previously shown to exert potent anti-T. cruzi activities compared to the reference drug, benznidazole [50 value, followed by GT5A, whereas compound GT5B had the lowest EC50 value . Incubation with ET-1 was associated with signs of cell hypertrophy Figures A and B, T. cruzi compounds benznidazole, BA5, GT5A, and GT5B 48 hours following treatment in different concentrations . Different parameters were combined, and principal component analyses were conducted, defining a cytoskeleton score and a mitochondrial score  are also safe and effective in cardiomyocytes brings valuable data for further development.hiPSC-CMs hold the potential to contribute to the anti-50 value of 5.9 μM, which is situated within the range of EC50 values reported in the literature for benznidazole in other cell types [50 of 3.2 μM for reducing T. cruzi amastigotes in hiPSC-CMs, half of the EC50 value found for the reference drug, benznidazole. Previously, an EC50 value of 1.8 μM was found for murine macrophages [T. cruzi drugs [50 values in hiPSC-CMs of 1.9 and 0.8 μM values, respectively, much lower compared to benznidazole. A previous study in macrophages reported higher values of EC50 for GT5A and GT5B, with 4.2 and 2.9 μM, respectively [Treatment of infected hiPSC-CMs with the reference drug benznidazole resulted in an ECll types , 18, 19.ll types . In our rophages . Thiazolzi drugs . Both GTzi drugs . These cectively .50 of 37 μM, which is discrepant and significantly lower than the values previously reported for murine macrophages [Our data also revealed cell type-specific toxicities for some of the compounds, which were not reported in previous studies with murine cells. BA5 showed lethal toxicity for hiPSC-CMs with a CCrophages . At loweThe mechanical stability of the cardiomyocyte depends on the integrity of the cytoskeleton. Geometric changes in the cell membrane can lead to changes in electrophysiology . Some drDysfunctional mitochondria can compromise myocardial function , 27 as cT. cruzi activity but also less toxic to hiPSC-CM, with less altered cell morphology, as demonstrated by multiparametric HCS analysis.The compounds tested herein comprehend the two main classes of compounds currently explored as antiparasitic agents for Chagas disease: molecules with exclusive antiparasitic activity (GT5A and GT5B) and molecules with dual antiparasitic and anti-inflammatory/immunomodulatory activity (BA5). Another clinical significance of the compounds is their pharmacological profile. In cardiomyocytes, we found that all three compounds have IC50 values lower than the reference drug benznidazole, making them candidates for further development. However, the toxicity analyses showed that BA5 may present a cardiotoxicity profile, an observation similar to a high concentration of GT5A. But unlike GT5A, GT5B was not only the most potent compound in terms of anti-A limitation of the present study involves the degree of maturity of hiPSC-CMs. So far, hiPSC-CMs that have been used for the cardiotoxicity test show a structural phenotype compatible with fetal cardiomyocytes. The stage of development of hiPSC-CMs used in this study is in accordance with the literature, which reports that cardiac differentiation from hiPSC routinely leads to the generation of cells with an immature structural and functional phenotype, of the fetal type . HoweverT. cruzi activity in hiPSC-CMs, using multiparametric analyses on a high-content screening platform. This assay was able to confirm the anti-T. cruzi activity of BA5, GT5A, and GT5B and identified the compound GT5B as promising, due to its potency and low toxicity in cardiomyocytes. In addition to having the advantage of being based on the use of human cardiac cells affected by Chagas disease, the assay has the advantage of allowing the rapid assessment of anti-T. cruzi actions in addition to parameters of lethal and sublethal cardiotoxicity, which can increase the predictive value of the tests.The use of hiPSC-CMs in the drug development workflow for Chagas disease has the potential to assist in the identification of new compounds and to predict cardiotoxicity. In this work, we established the test for infection and screening compounds with anti-


The first descriptions of reinfection by SARS-CoV-2 have been recently reported. However, these studies focus exclusively on the reinfected case, without considering the epidemiological context of the event. Our objectives were to perform a complete analysis of the sequential infections and community transmission events around a SARS-CoV-2 reinfection, including the infection events preceding it, the exposure, and subsequent transmissions. Our analysis was supported by host genetics, viral whole-genome sequencing, phylogenomic viral population analysis, and refined epidemiological data obtained from interviews with the involved subjects. The reinfection involved a 53-year-old woman with asthma (Case A), with a first COVID-19 episode in April 2020 and a much more severe second episode 4-1/2 months later, with SARS-CoV-2 seroconversion in August, that required hospital admission. An extended genomic analysis allowed us to demonstrate that the strain involved in Case A’s reinfection was circulating in the epidemiological context of Case A and was also transmitted subsequently from Case A to her family context. The reinfection was also supported by a phylogenetic analysis, including 348 strains from Madrid, which revealed that the strain involved in the reinfection was circulating by the time Case A suffered the second episode, August-September 2020, but absent at the time range corresponding to Case A’s first episode.IMPORTANCE We present the first complete analysis of the epidemiological scenario around a reinfection by SARS-CoV-2, more severe than the first episode, including three cases preceding the reinfection, the reinfected case per se, and the subsequent transmission to another seven cases. On 24 August 2020, the first SARS-CoV-2 reinfection case  was repo2–Prolonged SARS-CoV-2 RNA shedding may extend up to 101 days .per se. Here, we describe a SARS-CoV-2 reinfection case in a subject without clinical risk factors and analyze the epidemiological scenario before and after the reinfection. We identify the exposure event responsible for the reinfection and describe the extensive transmission from the reinfected case to her relatives.To date, reports on reinfection cases focus on the analysis of the case TC] value 30). Since the patient had no respiratory failure or other serious conditions, she was discharged from hospital the same day and given symptomatic treatment. The patient remained symptomatic for over 2 weeks (mainly dyspnea and fever) with eventual resolution of symptoms. On 18 April, a second reverse transcriptase PCR (RT-PCR) was performed with a positive result. One month after discharge, the SARS-CoV-2 RT-PCR was repeated (12 May), and this time the result was negative. SARS-CoV-2 serology was not available at that time.A 53-year-old woman (Case A) was admitted to the emergency room in our hospital  on 3 April 2020 presenting with dyspnea, fever, cough with expectoration (24 h), and a history of bronchial asthma. She had been treated regularly with inhaled budesonide, with no systemic corticosteroids between April and August 2020. At admission, HIV serology and IgG, IgM, and IgA determinations were requested with negative and normal results, respectively. Blood tests and chest X ray showed no outstanding changes. Positive SARS-CoV-2 PCR was obtained for the nasopharyngeal exudate .On 14 August, 4-1/2 months (140 days) from her first positive RT-PCR, Case A began to have fever, dyspnea, cough, and arthromyalgia. Another SARS-CoV-2 RT-PCR was performed (21 August) with a positive result  and C-reactive protein (CRP) stood out.TC values 21, 33, and 33, respectively). The patient progressively improved and was discharged 17 days after admission with oxygen therapy. Prior to discharge, the SARS-CoV-2 serology  was repeated (8/9). The result was positive with titers of 7.04.During the first 48 h, Case A showed radiological worsening and respiratory failure, experiencing significant bronchospasm. She was given corticosteroids, remdesivir, and lopinavir/ritonavir. Three RT-PCRs were performed during hospitalization . His symptoms resolved on 18 August.Twelve days before (7 August) the onset of case A’s second episode, she had had close physical contact with an uncle . BesideCase A was interviewed for more details regarding her and her uncle’s epidemiological context. Three of her uncle’s friends had also fallen ill with COVID-19; all four attended the same religious center every Friday and had had additional contacts. When interviewed, one of them said that the last time all four met was at the religious center on 7 August. The three friends had subsequent positive RT-PCRs . Case ATC value 25). On 28 August, his dyspnea worsened and an interstitial infiltrate was observed in the upper left lobe. Home isolation was indicated for 14 days.On 25 August, 11 days after the onset of Case A’s second episode symptoms, her husband complained of fever and malaise. He tested positive for SARS-CoV-2 by PCR . Home isolation ended after 14 days, without RT-PCR control. The daughter’s husband began having symptoms the same day (18 August) and tested positive on 21 August (RT-PCR) and 2 September. The daughter’s husband had contact only with his wife and not with case A . The RT-We first confirmed that the specimens isolated from Case A, who had had positive SARS-CoV-2 RT-PCR results in April (first episode) and August (second episode) belonged to the same patient, as indicated by the identical microsatellite short tandem repeat (STR)-PCR patterns obtained from the human DNA in the corresponding samples  to determine the complete consensus sequence and single-nucleotide polymorphism (SNP) calling with high confidence. WGS analysis of the specimens from episode 2 provided good coverage . We determined 16 SNPs using the Wuhan-1 sequence as reference (seven of which were missense variants)  and assiWe extended the genomic analysis beyond Case A and included (i) the strain from the potential source of Case A’s second episode, her uncle, and (ii) the strains from two of her uncle’s friends (uncle’s exposure context). Moreover, we included the strain identified in Case A’s husband . No samples for sequencing were available from Case A’s daughter, her son-in-law, or her granddaughter. The four studied specimens yielded sequences of enough quality  to allow comparisons throughout the whole genome. Case A and her husband had identical sequences and differed from those of case A’s uncle and his friends in 1 SNP . This inhttps://nextstrain.org/) shows that the strain identified in the second episode belongs to a clade, 20A.EU1, that had not been described in GISAID before the end of June 2020 , and the five cases in this study shared a single proper branch within this clade . The strune 2020 .10.1128/mSphere.00596-21.2FIG S2FIG S2, PDF file, 0.1 MB.Dated phylogeny of the strains closely related to that identified in Case A’s second episode. The strain identified in Case A is marked with a black arrow and belongs to a phylogenetic clade, 20A.EU1, which was not circulating before the end of June 2020, ruling out its presence in the first wave. Download Copyright © 2021 Pérez Lago et al.2021Pérez Lago et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Reinfection as the most likely explanation for Case A’s second episode is based on the following: (i) 4-1/2 months between her first and second episodes and confirmation by STR analysis that the specimens from the two RT-PCR-positive SARS-CoV-2 episodes came from the same patient; (ii) negative PCR after the resolution of the first episode; (iii) COVID-19 antibody seroconversion in Case A in the second episode; (iv) close physical contact without protective measures with a family member (uncle) who began with symptoms 2 days after their encounter, had a positive RT-PCR 5 days later, and started with symptoms 5 days before the onset of Case A’s symptoms in her second episode; (v) identification by WGS that the SARS-CoV-2 strains from Case A and her uncle were almost identical (1 SNP); and (vi) determination that the strain was circulating in her uncle’s epidemiological close context and in Madrid during the second episode but not at the time of the first episode.There was no evidence of immunosuppression in Case A. She received only regular treatment for her asthma with no systemic corticosteroids. A reinfection case resembling the one in this study was reported in Belgium: female, immunocompetent, similar age (51 years), and asthma as the only risk factor, for which she was given oral corticosteroids .Time lapses between the first and second episodes in previously reported COVID-19 reinfection cases 15, 12–15 12–To date, there is no defined pattern of severity for second episodes following reinfection. Some reinfection cases have been reported to be asymptomatic , 12, whiIn most reported reinfection cases, SARS-CoV-2 IgG serology is not well documented for the first episodes because during that time (March-April 2020) there was limited availability of tests. This implies that in some reinfections with seroconversion in the second episode, as in our case, it is not possible to determine whether the case did not mount an immune response in the first episode or whether antibodies were lost before the second episode was diagnosed. Similar uncertainties have been described for SARS-CoV-2 reinfections elsewhere , 2.Genomics is the ultimate support to document reinfections. The most obvious way to determine a reinfection is to compare the strains from each episode. Unfortunately, and similarly to other COVID-19 reinfection reports , we coulThe European Centre for Disease Prevention and Control has accepted that reinfections may be documented either by identifying genomic differences between the two episodes or by confirming that the strain from the second episode clusters with strains from the location of exposure . Thus, wUnderstanding Case A’s epidemiological context allows us to show for the first time the onward transmission leading to a reinfection and to report subsequent transmission from a reinfection case. The identity of Case A and her husband’s strains, including a private marker SNP not shared by other cases, the chronology of symptom onset, and the confirmation that Case A’s husband did not interact with her uncle or his friends and did not attend the religious center confirmed Case A as the sole source for the infection of her husband.There was a likely noteworthy transmission from Case A after her reinfection, beyond her husband, deduced from the sequential chronology of symptoms and positive RT-PCRs obtained for her daughter, son-in-law, and four grandchildren. Unfortunately, no samples were available from these subjects as they were diagnosed in another institution where specimens were not stored. Therefore, the lack of sequencing data from the son-in-law and grandchildren impeded us from fully supporting their involvement in the transmission chain.Here, we describe the first complete analysis of the epidemiological scenario around a reinfection by SARS-CoV-2 supported by host genetic analysis, viral genomic analysis, phylogenomic population analysis, and in-depth epidemiological investigation. This extensive approach documents a much more severe second COVID-19 episode in a woman without risk factors, except for asthma, after exposure through a close contact to a SARS-CoV-2 strain actively transmitted in the same setting/population. Once reinfected, our case was responsible for an extensive transmission among family members.The study was approved by the ethical research committee of Gregorio Marañón Hospital (REF: MICRO.HGUGM.2020-042). Informed consent was obtained from the patient for publication of this case report.The remnants from diagnostic nasopharyngeal SARS-CoV-2 RT-PCR-positive swabs were aliquoted and stored at −70°C until analysis.RNA was extracted and purified from 300 μl of nasopharyngeal exudates using the EasyMag  (specimen from April) or KingFisher  (specimens from August-September) instruments.Next, real-time RT-PCRs were performed. The novel coronavirus (2019-nCoV) nucleic acid diagnostic kit  was used for the April specimen, and the TaqPath COVID-19 CE-IVD RT-PCR kit  was used for the August-September specimens.Eleven microliters of RNA was used as the template for reverse transcription with Invitrogen SuperScript IV reverse transcriptase  and random hexamers . Whole-genome amplification of the coronavirus was done with the Artic_nCov-2019_V3 panel of primers  (artic.network/ncov-2019) and the Q5 Hot Start DNA polymerase enzyme . Libraries were prepared using the Nextera Flex DNA library preparation kit , following the manufacturer’s instructions.Libraries were quantified with the Quantus fluorometer  before being pooled at an equimolar concentration (4 nM). Next, libraries were sequenced in pools of up to 17 libraries on the MiSeq system  using the MiSeq Reagent Micro kit v2 (2 × 151 bp) or in pools of up to 96 libraries with the MiSeq reagent (2 × 201 bp).https://gitlab.com/fisabio-ngs/sars-cov2-mapping. Briefly, the pipeline goes through the following steps: (i) removal of human reads with Kraken  prh Kraken  v 0.20.1https://zenodo.org/record/4069557#.X37nuXUzaWg). Clade nomenclature was assigned using Nextstrain  as recommended by the manufacturer.Short tandem repeat (STR)-PCR  was applied for the human identity testing analysis. For this, the specimens used for SARS-CoV-2 genome sequencing were employed. We examined 12 noncoding STR loci and the gender-specific locus amelogenin see . PCR was10.1128/mSphere.00596-21.3TABLE S1Table S1, DOCX file, 0.02 MB.Noncoding STR loci and the gender-specific locus amelogenin used for the short tandem repeat analysis. Download Copyright © 2021 Pérez Lago et al.2021Pérez Lago et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://www.ebi.ac.uk/ena/browser/home) .The data that support the findings of this study (Fastq files) are publicly available. Fastq files above the GISAID thresholds were deposited at GISAID . All sequences were also deposited at the ENA (European Nucleotide Archive;


Objectives: To assess the efficacy of intralesional injection of mitomycin C (MMC), using a novel adjustable-tip needle, following visual internal urethrotomy (VIU) in reducing the recurrence of de novo bulbar urethral stricture disease (USD).Patients and methods: Using closed envelopes, 50 patients diagnosed with bulbar USD of <2 cm were randomised to undergo either VIU with MMC injections (Group-A) or VIU alone (Group-B). The urethrotomy was performed under direct vision using a cold-knife and incisions were made at the 12-, 4-, and 8-o’clock positions followed by intralesional injection of 10 mL MMC (0.4 mg/mL) using a novel depth-adjusting needle. All patients were objectively evaluated pre- and postoperatively at 3, 6, and 9 months using uroflowmetry (maximum urinary flow rate), post-void residual urine volume, and retrograde urethrography.Results: Of all 50 patients; five missed follow-up , hence 45 cases were available for analysis (21 in Group-A and 24 in Group-B). The mean (SD) age of patients was 34.33 (7.2) and 37.7 (10.2) years in Group-A and Group-B, respectively (P = 0.22). The depth-adjusting needle was easy to use and all injections were successfully completed. In terms of stricture recurrence, there was significant decrease in Group-A  compared with Group-B;  (P = 0.01). On multivariate Cox regression analysis, the VIU with MMC was found as a sole factor associated with marked decrease in stricture recurrence . The Kaplan–Meier survival curve for recurrence-free survival showed a statistically significant difference between both groups .Conclusion: The use of a novel depth-adjusting needle was easily applied and MMC injection after VIU resulted in a significantly lower recurrence of de novo bulbar USD.Abbreviations : MMC: mitomycin C; PVR: post-voiding residual urine; Qmax: maximum urinary flow rate; RFS: recurrence-free survival; RUG: retrograde urethrography; USD: urethral stricture disease; VIU: visual internal urethrotomy Urethral stricture disease (USD) is a term that refers to the scarring process involving the epithelium or spongy erectile tissue of the corpus spongiosum (spongiofibrosis); therefore, it is considered as an anterior urethral disease. USD can be asymptomatic for a while, but because the lumen is further reduced, it can be associated with marked voiding symptoms . There aThe VIU is one of the basic methods for the treatment of USD. Being a minimally invasive procedure with less morbidity, it is commonly performed by endourologists around the globe . Successde novo bulbar USD. Our hypothesis was that appropriate intralesional injection of MMC by a special adjustable-tip needle after subjective estimation of the depth of injection, as an adjunctive procedure, can unify the depth of injection along the cohort which undergo injection and make the procedure easier and more successful, the results more reliable, and significantly reduce recurrence of USD after VIU.MMC is a chemotherapeutic agent that has potent alkylating anti-neoplastic antibiotic activity by inhibition of DNA synthesis by cross-linking DNA between guanine and adenine, and suppression of cellular RNA and protein production. Hence, it prevents replication of fibroblasts and epithelial cells and inhibits collagen synthesis resulting in delayed wound contraction ,7. The Mmax) of <12 mL/s. Patients with complex stricture, complicated by a fistula or abscess, blind urethral stricture, history of urethroplasty, and full-thickness spongiofibrosis were excluded. Patients who agreed to participate in this study were randomised into two groups: Group-A (study group) underwent intralesional MMC after VIU and Group-B (control group) underwent VIU only (This randomised controlled study was conducted in our tertiary care urology institute between April 2018 and July 2020 (including the follow-up period), after obtaining ethics approval and informed consents. Randomisation was achieved using closed envelopes. We recruited all patients with single, bulbar USD of a maximum length of 2 cm, with partial thickness spongiofibrosis on sonourethrography and a maximum urinary flow rate  is applied to identify the non-distensible narrow area and measure it [All patients were evaluated preoperatively based on full history, clinical examination, routine laboratory investigations, retrograde urethrography (RUG), abdomino-pelvic ultrasound with estimation of post-voiding residual urine (PVR) volume, uroflowmetry to assess Qasure it .The intervention was performed under spinal anaesthesia. Preoperative antibiotic prophylaxis with a single oral dose of levofloxacin 500 mg was given. Patients were put in lithotomy position, and cysto-urethroscopy was performed using a 17-F rigid cystoscope to allow for a guidewire and ureteric catheter to pass through the stricture into the urinary bladder. Cold-knife incisions, at the 12-, 4-, and 8-o’clock positions, were made through the whole thickness of the fibrosis until healthier tissue appeared . In GrouThe depth of injection was subjectively estimated during VIU and the required depth was adjusted  . The patmax. All patients were followed-up at 3, 6 and 9 months for clinical assessment of obstructive symptoms, Qmax, PVR by abdomino-pelvic ultrasonography, and RUG. The main outcome parameter was urethral stricture recurrence, which was identified as recurrence of LUTS during follow-up visits starting at 3 months with difficulty in passing a 16-F urethral catheter and confirmed by RUG [All patients were asked to attend at 1 month for assessment of obstructive symptoms and undergo abdomino-pelvic ultrasonography for calculation of PVR and undergo uroflowmetry for assessment of voiding pattern and calculate Qd by RUG .t-test of significance was used when comparing between two means, and the paired sample t-test was used to compare the outcomes within groups at 3, 6, and 9 months. The chi-square test of significance was used to compare proportions between two qualitative parameters. A two-tailed P ≤ 0.05 was considered significant. The Kaplan–Meier survival curve was used to track recurrence-free survival (RFS). Factors affecting RFS were compared at the univariate level using the log-rank test. Multivariate stepwise Cox regression analysis was used for identification of predictors of recurrence. Hazard ratios (HRs) with 95% CIs were calculated for predictors.Sample size was calculated using the G*Power 3.1.9.7 for Windows. Based on alpha adjusted at 0.05 and the power adjusted at 80%, and an expected difference in recurrence rate of 40% between Group-A (VIU with MMC injection) and Group-B (VIU only) [From a total of 50 randomised patients (25 in each group), five patients (10%) discontinued the study; four of 25 (16%) and one of 25 (4%) patients were lost to follow-up in groups A and B, respectively. Overall, 45 patients were available for analysis; Group-A, 21 patients and Group-B, 24 . All patmax was 8.69 (1.55) mL/s and the preoperative PVR was 76 (27.45) mL.The mean (SD) age of all patients was 36.13 (9.03) years and the mean (SD) length of the stricture was 1.31 (0.40) cm, the majority of patients (35 patients) had partial thickness spongiofibrosis and only 10 patients had no spongiofibrosis. In terms of the cause of stricture, an idiopathic origin was the most common cause (36 patients), the mean (SD) preoperative QP= 0.22), cause of stricture (P= 0.71), and length of stricture  and PVR  and postoperative complications (P= 0.40); bleeding 9.5% in Group-A (two patients) and 8.3% in Group-B (two), and postoperative fever (only two in Group A and three in Group B). Nevertheless, there was highly statistically significant difference between pre- and postoperative Qmax and PVR between the two groups  for injecting MMC, and assessed the effect of this on the recurrence of USD. Of the 50 patients recruited for the present study, four patients in Group-A and one in Group-B did not attend for follow-up in the first 3 months; hence, were dropped from the study. The sample during analysis was thus reduced to 45 patients, Group-A (study group) comprised 21 patients who had intralesional MMC after VIU and Group B (control group) comprised 24 patients who underwent VIU only.et al. [et al. [et al. [et al. [et al. [et al. [Regarding the demographic assessment of our patients, the mean (SD) age in Group-A was 34.33 (7.23) years, while in Group-B it was 37.71 10.26) years. This was consistent with the age group of the Ali  years. T [et al.  operatedmax was 9.0 (1.73) mL/s in Group-A and 8.42 (1.35) mL/s in Group-B, while the mean (SD) Qmax at 9 months was 17.43 (3.26) mL/s in Group-A and 13.63 (3.49) mL/s in Group-B, which was highly statistically significant. These results were congruent with the study of Farrell et al. [max on uroflowmetry was significantly increased after VIU with MMC .In the present study, the preoperative mean (SD) Qet al. [P= 0.22). The authors considered this to be due to the relatively low preoperative PVR.In addition to the above results, there was a statistically significant decrease in PVR at 9-months postoperatively compared with the preoperative measurements in both groups A and B, at a mean (SD) of 29.90 (23.12) vs 62.71 25.89) mL and 58.75 (38.23) vs 87.62 (23.59) mL, respectively. However, the results from the study of Farrell 9 mL and et al. [Regarding complications, the present study found that there was no statistically significant difference between the two studied groups for intra- and postoperative complications . There was also no significant difference between the two groups in the study by Ali et al. .P= 0.01). These recurrence rates were comparable with the study by Madzok et al. [et al. [et al. [et al. [P= 0.04).In terms of stricture recurrence in our present study, there was significant decrease in Group-A  compared with Group-B  (.3%, one Limitations of the present study include the relatively short period of follow-up. However, USD recurrence usually occurs in the first 3–6 months. Another limitation is that all patients were followed-up by RUG because we do not have flexible ureteroscopy on site. Also, patients without spongiofibrosis on sonourethrography were not randomised between the two groups. Additionally, we did not plan to assess the safety of MMC injection following VIU. However, no adverse events related to MMC injection were identified during the follow-up period. Nevertheless, the present study is the first to use a novel depth-adjusting needle for intralesional injection of MMC after VIU and this study adds to the scant literature about the efficacy of intralesional MMC injection following VIU of bulbar USD.de novo bulbar USD. Other multicentre prospective studies recruiting more patients are encouraged.The use of a novel depth-adjusting needle was easily applied for MMC injection after VIU and MMC injection after VIU resulted in a significantly lower recurrence of


Additionally, diffusion was denoted as a limiting step in the irreversible electrooxidation process that occurs in the proposed system.The diversity of materials proposed for non-enzymatic glucose detection and the lack of standardized protocols for assessing sensor performance have caused considerable confusion in the field. Therefore, methods for pre-evaluation of working electrodes, which will enable their conscious design, are currently intensively sought. Our approach involved comprehensive morphologic and structural characterization of copper sulfides as well as drop-casted suspensions based on three different polymers—cationic chitosan, anionic Nafion, and nonionic polyvinylpyrrolidone (PVP). For this purpose, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy were applied. Subsequently, comparative studies of electrochemical properties of bare glassy carbon electrode (GCE), polymer- and copper sulfides/polymer-modified GCEs were performed using electrochemical impedance spectroscopy (EIS) and voltammetry. The results from EIS provided an explanation for the enhanced analytical performance of Cu-PVP/GCE over chitosan- and Nafion-based electrodes. Moreover, it was found that the pH of the electrolyte significantly affects the electrocatalytic behavior of copper sulfides, indicating the importance of OH The most practical and thus commercialized methods for the self-monitoring of diabetes are based on the electrochemical measurement of glucose levels. Classical sensing setups utilize enzymes, glucose oxidase, or glucose dehydrogenase. The first generation of enzymatic sensors is based on the measurement of hydrogen peroxide produced during enzyme-catalyzed glucose oxidation. However, the dependence on oxygen presence in the system severely hinders their reliability, which has become an incentive for the development of the next generation in which a mediator  is responsible for electron transfer. Here, the major drawback is the interference effect of other electroactive species. A real breakthrough in point-of-care diagnosis was the proposal of direct electron transfer implemented by connecting electrically active enzyme sites with the electrode ,2. It alNon-enzymatic glucose oxidation has been the subject of intensive studies for almost 40 years. The first stage of research, dating back to 1982, concerned understanding the nature of glucose electrochemistry on platinum electrodes . Howeverads) forms. It mediates glucose oxidation, but at the same time, inhibits reduction processes ), potassium ferrocyanide (K4[Fe(CN)6]), acetic acid , sulfuric acid , and sodium hydroxide solution  were obtained from Avantor . Polyvinylpyrrolidone  and Nafion D-520 dispersion (5 wt.%) were supplied by Alfa Aesar . The chemicals were analytical grade and were used without additional purification. Aqueous solutions of 1 M glucose, 25% acetic acid, 0.1 M KCl + 0.2 mM [Fe(CN)6]3−/4−, and 0.1 M KCl + 1 mM [Fe(CN)6]3− were prepared with deionized water. Silver chloride electrodes , platinum wire (diameter of 0.7 mm), and glassy carbon electrodes  were manufactured and provided by Mineral Company . The glassy carbon used in the GCEs was produced during the two-step pyrolysis of highly cross-linked furan-based epoxy resin at a maximum temperature of 2200 °C (type G).Low molecular weight chitosan and phosphate-buffered saline  were purchased from Sigma-Aldrich . Anhydrous copper chloride . The synthesis temperature was set to 200 °C and time to 6 h. Afterward, the autoclave was cooled to room temperature naturally. The obtained product was washed with a water/ethanol solution (1:1 volume ratio), centrifuged, and dried in a vacuum oven at 60 °C for 12 h.Copper sulfide microstructures were obtained according to a previously reported procedure . In the −1 at 488 nm excitation wavelength. For the evaluation of the phase composition, X-ray diffraction (XRD) was utilized. Analysis was conducted by X’Pert MPD diffractometer  equipped with Johansson monochromator . The specific surface area of the powder was measured using the ASAP 2020 Plus analyzer .Scanning electron microscopy (SEM) was applied for the investigation of copper sulfide morphology. Images were obtained using a Nova NanoSEM 200 . The structure was determined by Raman spectroscopy performed on Raman confocal microscope alpha 300R . Spectra were collected in the range of 200–1600 cm2SO4 following the procedure proposed by Gao et al. 3− was used. The voltammograms were recorded at different scan rates (6.25–500 mV/s) in the potential range −100–1000 mV.All voltammetry experiments were conducted using an electrochemical analyzer M161E  in a three-electrode system with bare or modified GCE serving as a working electrode, silver chloride electrode  as a reference electrode, and platinum wire as an auxiliary electrode. To determine the electrochemically active surface area of the electrodes, the cyclic voltammetry (CV) technique was applied. As an electrolyte, a solution of 0.1 M KCl + 1 mM 3−/4− redox species (O/RD = 7.2 × 10−6 cm2/s).The heterogenic rate constant of the electrode reaction tion (3) :(3)k=WCTCTR value for PVP/GCE than for bare GCE can be explained by the formation of a dielectric shell 3−:I is the peak current [A], O/RD—diffusion coefficient of [Fe(CN)6]3−/4−—redox species (O/RD = 7.2 × 10−6 cm2 /s), n—number of electrons involved in the redox reaction (n = 1), 0C—concentration of redox species (0C = 1 mM), and v—scan rate [mV/s].The Randles-Sevcik equation was applied for the determination of the electrochemically active surface area (EASA) using cyclic voltammetry in 0.1 M KCl + 1 mM [Fe(CN)2 possessed chitosan-modified GCE . The lower EASA of bare GCE than its geometric area (7.07 mm2) can be attributed to the applied activation procedure and changes introduced in the surface composition and roughness [2), while the calculated values for Cu-NAF/GCE and Cu-CH/GCE were comparable . This was attributed to the most uniform coverage with particles of the substrate electrode ensured by PVP, which provided steric stabilization of copper sulfides within the prepared suspension and drying droplet. Details on stabilization mechanisms provided by different polymers have previously been reported [Analysis of the results indicated that the highest EASA of 50.98 mmfied GCE . This maobserved . These ooughness ,35. The reported .The first step in evaluating the glucose-sensing abilities of the modified electrodes involved the determination of the glucose oxidation potential by means of CV. For this purpose, cyclic voltammograms were recorded after subsequent glucose addition in the concentration range 0–1.5 mM. As can be seen in oxE = 680 mV) compared to other electrodes (oxE = 590 mV) was detected. These observations are in good agreement with results from EIS, which indicated preferable electrochemical properties of Cu-PVP/GCE (low charge transfer resistance and high heterogeneous rate constant of the electrode reaction) over chitosan- and Nafion-based electrodes.Moreover, for the latter, also a shift in the position of the oxidation peak (2 = 0.996) and 0.42 (R2 = 0.997), respectively, which is relatively close to the expected theoretical value for the diffusion-controlled process (0.5). The slope for Cu-CH/GCE was almost two times lower . This may be explained by the characteristic sigmoidal shape of the voltammogram for this electrode, indicating the occurrence of a multiplied microelectrode effect [3O4) developed by Ding et al., a surface-limited detection process occurred [To determine whether glucose oxidation by modified electrodes is a diffusion- or surface-controlled process, voltammograms were recorded at different scan rates in the electrolyte containing 1 mM glucose. Subsequently, the logarithmic dependence of the scan rate on the generated peak current was plotted b. The obe effect . Formed e effect . This mee effect . On the occurred . Furtheroccurred , electroads suggested by the Incipient Hydrous Oxide-Adatom Mediator (IHOAM) theory [j) for core-shell CuS-Cu2S decorated carbon nanotube-graphene composites [j was obtained at pH = 12.7, while a further increase of pH resulted in a signal drop, which is in agreement with our results. Moreover, the same behavior was observed in the case of electrodes working at neutral pH developed by Gao et al. [2-CuS/GCE electrodes were tested in PBS with different pH values ranging from 5.5 to 8.0. The highest peak current was obtained at pH = 7.2.Another investigated issue was the influence of the electrolyte pH on the signal obtained from the glucose. In most articles devoted to non-enzymatic glucose sensors based on copper sulfides, an alkaline medium was used for the measurements . This is) theory . It was ) theory . In anotmposites . It was o et al. . In thei1.8S/CuS modified electrodes was adapted:Based on the findings of Burke, subsequent studies on glucose oxidation via transition metals ,43,44,45Glucose detection was performed by chronoamperometry. Appropriate calibration curves were prepared in the range of 0–1 mM (step 0.1 mM). The limits of detection (LOD) and quantification (LOQ) were evaluated using the signal-to-noise (S/N) method with S/N ratios of 3 and 10, respectively. Sensitivity was calculated based on the determined slopes and EASA. The assessed values are listed in CTCPE compared to that of other electrodes [All samples exhibited comparable sensitivity; however, generated signals differed significantly. This may be attributed to the available active sites of copper sulfides. In the case of chitosan- and Nafion-based electrodes, these polymers blocked some of them, as also indicated by decreased values of EASA and higher Warburg impedance modulus ,46. The ectrodes .I) values over a wide range of concentrations (glucoseC = 0.01–20 mM). It was found that in the very limited range, the current dependency on glucoseC is linear, whereas altogether, it follows a Langmuir isotherm  of the proposed fittings, are summarized in aCglucose << 1K), Equation (8) can be simplified to glucoseI = KC, which is in good agreement with the evaluated slopes of the calibration curves. Ding et al. related the applicability of this approach to surface catalytic reactions [2 > 0.999) was evaluated to be 0.06–1.00 mM for Cu-CH/GCE, 0.06–1.50 mM for Cu-NAF/GCE, and 0.02–2.00 mM for Cu-PVP/GCE.The determined values of eactions . The obseactions . The linAdditionally, the repeatability and reproducibility of the investigated electrodes were evaluated. The repeatability test consisted of comparing the responses of the same electrode to 0.5 mM glucose addition during three independent measurements. The calculated relative standard errors (RSE) were equal to 4.4%, 3.1%, and 3.2% for Cu-CH/GCE, Cu-NAF/GCE, and Cu-PVP/GCE, respectively, indicating satisfactory repeatability. To determine reproducibility, three different electrodes were fabricated, and calibration curves in the glucose concentration range 0–1 mM were prepared using them . The RSEThis work underlines crucial steps that should be undertaken when evaluating the impact of the single working electrode component on the sensor analytical performance. We proposed a double-track approach. The first considered aspect included evaluation of the morphology and structure of the modifying layers, with special emphasis on the distribution of particles on the surface. Another important issue was the determination of changes brought by each modifying component to the electrode electrochemical properties, which was achieved efficiently by utilizing electrochemical impedance spectroscopy. The information provided by this technique combined with microscope observations allowed us to predict the analytical performance of modified electrodes. In our case, the application of PVP as a binder in drop-casted suspension ensured both uniform coverage of the GCE surface with copper sulfides and superior electrochemical properties in comparison with chitosan- and Nafion-based electrodes. We also conducted comprehensive studies on the influence of electrolyte pH on the Cu-PVP/GCE response using cyclic voltammetry. This technique also served to determine the limiting process during detection. Nonetheless, further extension of the EIS measurements is required to gain a better insight into the electrocatalytic activity of copper sulfides in glucose oxidation. This includes measurements in electrolytes covering a wider range of pH values and containing different glucose additions, as well as conducting experiments in a three-electrode system at the applied potential.


Interestingly, the pseudo-first-order reaction rate of a Povarov model reaction catalyzed by PAF-201 as a Lewis acid was ∼3.7 times and ∼4.7 times as those of tritylium tetrafluoroborate and tri(4-biphenyl)carbonium tetrafluoroborate, respectively. Theoretical calculations revealed that the tritylium ion at the node of PAF-201 has a quasi-planar structure. The transformation of triphenylcarbinol in PAF-200 to tritylium in PAF-201 can make the framework taut, and the rebounding force toward the tetrahedral structure is stored. This is favorable for tritylium to activate the imine substrate along with a deformation of the quasi-plane to tetrahedron. PAF-201 could be easily recycled at least three times without evident loss of catalytic activity. This work presents the catalytic activity of the tritylium ion under stress.Tritylium salts have been used as Lewis acid catalysts in organic synthesis for a long time. In this work, we found that the Lewis acid catalytic activity of tritylium ions at the node of a tensile framework is significantly improved compared to that of the free tritylium salts. The tritylium-based framework, PAF-201 , was prepared by acidification of a semi-rigid triphenylcarbinol-based parent framework, PAF-200. When PAF-200 was alternately exposed to HCl and NH In this work, a tensile tritylium-based organic framework, PAF-201 was prepared. The Lewis acid catalytic ability of PAF-201 was significantly higher than that of tritylium tetrafluoroborate. This work presented the catalytic activity of the tritylium ion under stress. In addition, research on porous ionic polymers has mainly focused on boron-,8 nitrogen-,9 oxygen-10 or phosphorus-centered ions,11 but seldom on carbon-centered ions by now.12Porous organic polymers (POPs) have been developed rapidly owing to their significant advantages, such as large specific surface area and high stability.13 However, their use is limited by their high price and difficult recovery. Notably, porous aromatic frameworks (PAFs) have high-porosity structures, chemical affinities that can be tuned through the choice of node and linker, and extraordinary hydrothermal stability and chemical compositions.14 In this work, we plan to introduce tritylium units into the PAF framework so as to obtain an easily recycled material with the properties of tritylium salts. Considering that tritylium salts are generally prepared by acidification of triphenylcarbinol, it is a simple idea to use a triphenylcarbinol-based framework as the precursor for a tritylium-based one. Notably, the tritylium ion is the most stable carbocation with a propeller-like conformation, and partial charge of the central carbenium is dispersed on three phenyl rings. There will be a big deformation from triphenylcarbinol to tritylium. Thus, it is necessary for the parent framework to withdraw the tension due to this deformation. Some triphenylcarbinol-based porous materials have been reported,15–16 but the corresponding tritylium-based frameworks as Lewis acids have not been introduced so far. Herein, the tritylium-based framework was first prepared. To our surprise, it was found that the tritylium ions at the node of the tensile framework exhibited better Lewis acid catalytic ability than the free ones.As we know, tritylium salts are widely used in organic reactions, especially as Lewis acid catalysts and dehydrogenative reagents.3-activated Friedel–Crafts reaction in CHCl3. The resultant material exhibited very low porosity . In more detail, the mesoporous size distribution of PAF-201 was evidently different from that of PAF-200, also suggesting the occurrence of framework deformation. In addition, we also prepared another triphenylcarbinol-based material (TAF–OH) by reductive coupling of tri(4-bromobenzene)carbinol, as reported previously by Comotti et al.16 However, this framework could not be transformed into the tritylium-based one, possibly due to its rigidity.First, we tried to synthesize a porous framework using commercially available triphenylcarbinol as the substrate by AlCly Fig. S1, possibly Fig. S1. Subsequ−1 and a C–O band at 1069 cm−1  spectrum of PAF-200 exhibited an O–H band at 3500 cm1 Fig. S2, indicat Table S2. The Fri PAF-200 , and a s spectra  and S2†.3CN resulted in a strong adsorption at around 540 nm  carbonium can occur in acidic solution with a distinct color change. The addition of a small amount of conc. HCl to TBOH in CHm Fig. S6. Interesm Fig. S6, the colig. S64) . The cols Fig. S7. The abo17 In order to evaluate the Lewis acid catalytic performance of PAF-201, the Povarov reaction was selected as the model reaction. First, 2,3-dihydrofuran and benzylidene aniline were reacted in anhydrous tetrahydrofuran at room temperature with different PAF-201 loadings (4) and TBBF4 at 0.25 mol% only afforded isolated yields of 74% and 67% in three hours  at 1 mol% as a proton scavenger was added into the Povarov reaction system  can completely transform tropilidene into tropolium tetrafluoroborate in anhydrous CH3CN at 0 °C. However, this oxidation using PAF-201 was completed in twelve hours, and only two hours for TrBF4. It was time-assuming that all the active tritylium units on the framework were transformed into tritan ones with the big framework deformation. The solid-state 13C CP/MAS NMR spectrum of the recycled PAF-201 sample showed the strengthened signal at 55 ppm, indicating that all carboniums were converted into C–Hs carbonium and tritylium units in the optimization. Thus, two cluster models, fixed tri(4-biphenyl)carbonium and tritylium, were used to represent the catalytic site of PAF-201. Their construction details are described in the ESI. As observed from via Lewis acidic tritylium ion to form intermediate INT1; (2) the [4 + 2] cycloaddition reaction between the activated INT1 and the electron-rich alkene 2,3-dihydrofuran with the concerted or step-wise pathways; (3) the dissociation of the cycloaddition product INT4 and the regeneration of the carbonium; and (4) the tautomerization  of the released product PRO. The free energy barrier of the fixed case (representing PAF-201) in each step of the Povarov reaction is lower than those for the free cases. We also noted that the free energy barriers of the former three reaction steps in the fixed tri(4-biphenyl)carbonium are lower than those in the free tritylium by 1.9 kcal mol−1, 1.0 kcal mol−1 and 2.9 kcal mol−1, respectively. These calculated results suggest that PAF-201 is a better Lewis acid catalyst than free tritylium and tri(4-biphenyl)carbonium, in agreement with the pseudo-first-order reaction rates.To rationalize the rate acceleration of the Povarov reaction observed with PAF-201 relative to TrBF−1 higher than that of the free one, indicating the existence of tension in the fixed structure. Thus, the PAF-201 framework was taut so as to reserve the rebound force for reforming the tetrahedral structure. This is favourable for tritylium to activate benzylidene aniline in the Povarov reaction along with a deformation of the quasi-plane to a tetrahedron. In addition, the quasi-planar structure is closer to the TS1 structure with the ∠C3––C4 angle of 144.83° than the perfect one. This means that a smaller distortion energy is required from the reactant to the TS1 as activating benzylidene aniline. As a consequence, we could conclude that the quasi-planar tritylium ion at the node of the tensile PAF-201 framework had better Lewis acid catalytic activity.To gain deeper insight into the origin of the high Lewis acid catalytic activity of PAF-201, we focused more closely on the geometric details of tri(4-biphenyl)carbinol and free and fixed tri(4-biphenyl)carbonium . In free3 (2.5 g) was evacuated and inflated with Ar three times. Then CHCl3 (120 ml) was injected into the flask via a syringe, and this resultant system was stirred for 1 h at 60 °C. Furthermore, a solution of tri(4-biphenyl)carbinol (1.0 g) in CHCl3 (30 ml) was added into the flask. The colour of the reaction system immediately changed from light yellow to purple. The mixture was stirred for 24 h at 60 °C. The resulting dark blue powder was collected by filtration, subsequently washed with 1 M HCl solution and water repeatedly, further purified by Soxhlet extraction successively with methanol, THF and CH2Cl2 for 36 h, and then dried at 120 °C for 12 h under vacuum to give the earthy yellow powder product (1.2 g).A 250 ml round-bottomed flask with anhydrous AlCl4–Et2O  was injected into the flask via a syringe at 0 °C and this reaction system was kept at 25 °C for 24 h. The raw powder was collected by centrifugation, repeatedly washed with anhydrous THF, and dried for 12 h under vacuum at 25 °C, to give the dark blue powder product (2.0 g).A 100 ml round-bottomed flask with PAF-200 (2.0 g) was evacuated and inflated with Ar three times. HBFThe characterization details of the PAF materials are provided in the ESI.†3 gas. PAF-201 exhibited better Lewis acid catalytic activity than TrBF4 and TBBF4. The theoretical computational results showed that the quasi-planar tritylium ion as a node of a tensile network could more easily interact with the imine substrate and also dissociate with the product. PAF-201 as a Lewis acid catalyst could be well recycled for at least three times without evident loss of catalytic activity. Moreover, PAF-201 was also used as a dehydrogenative reagent in transforming tropilidene into tropolium. The further application of this tensile framework is underway in our laboratory.To summarize, we have successfully prepared two new porous aromatic frameworks, triphenylcarbinol-based PAF-200 and tritylium-based PAF-201. Some of the triphenylcarbinol units of PAF-200 were transformed into tritylium ions, affording the taut PAF-201 framework. A fast allochroic cycle was observed when PAF-200 was alternately exposed to HCl and NHSome data has been provided in ESI.Z. B. and G. Z. initiated and designed this work. Z. B. and F. C. wrote this paper. S. Z. conducted the synthesis and characterization of PAFs. J. Z. and F. C. conducted the computational work. Y. Z. conducted partial characterization work. X. Z. and S. W. revised this paper.There are no conflicts to declare.SC-012-D1SC02594E-s001SC-012-D1SC02594E-s002